Composition for cytocompatible, injectable, self-gelling chitosan solutions for encapsulating and delivering live cells or biologically active factors

Abstract

The present invention provides compositions and methods for tissue repair using a cytocompatible self-gelling cross-linked hydrogel. The composition comprises a biocompatible mixture of chitosan, bifunctional dialdehyde, and hydroxylated polymer, which can be used to immobilize or encapsulate viable cells, or bioactive substances. The method includes the process of mixing bioactive substances, live cells, and/or extracellular matrix components with a cross-linking solution comprising a bifunctional aldehyde-treated hydroxylated polymer such as hydroxyethyl cellulose. The cross-linking solution is then mixed homogenously with a neutral isotonic chitosan solution. The chitosan becomes cross-linked by the bifunctional aldehyde, while the cells are protected from potentially nocive effects of the aldehyde cross-linker by the hydroxylated polymer. The injectable solution retains cell viability and bioactivity, and immobilizes cells at the site of injection or delivery. Depending on the particular application, mixtures of chitosan and bifunctional dialdehyde may be employed. The injectable solution also liberates bioactive substances with controlled release kinetics from the site of injection.

1 . A composition for immobilizing and encapsulating viable and functional cells or bioactive substances comprising: a) a liquid polysaccharide solution of isotonic neutral chitosan; and b) a cross-linking solution consisting of a bifunctional or multifunctional, aldehyde or aldehyde-treated hydroxyl-containing polymer dissolved in physiological media.  
     
     
         2 . The composition of  claim 1 , wherein the cross-linking solution consists of a bifunctional or multifunctional cross-linker and a hydroxylated polymer of appropriate ratio and molecular mass such as to permit the hydroxylated polymer to remain liquid in solution.  
     
     
         3 . The composition of  claim 1 , where the cross-linking solution consists of glyoxal, or glyoxal-treated hydroxyethyl cellulose dissolved in a physiological medium.  
     
     
         4 . The composition of  claim 1 , wherein the composition comprises: a) 0.5 to 5.0% by weight chitosan, or chitosan derivative, or poly-amine containing polymer; and b) 0.01 to 5.0% by weight hydroxyethyl cellulose, wherein said solution form a gel between temperatures of 4 C and 42 C, said gel providing a physiological environment for maintaining viability of cells.  
     
     
         5 . The composition of  claim 4 , further comprising: 
 c) 0.0001-3% glyoxal.    
     
     
         6 . The composition of  claim 4 , wherein the composition forms a gel within seconds to several hours after mixing (a) and (b).  
     
     
         7 . The composition of  claim 5 , wherein the composition forms a gel within seconds to several hours after mixing (a), (b) and (c).  
     
     
         8 . The composition of  claim 4 , wherein the solution forms a gel between temperatures of 20 C and 42 C.  
     
     
         9 . The composition of  claim 1 , wherein the chitosan is dissolved in dilute acid and mixed with 1.0 to 2.5% by weight of a salt of polyol consisting of mono-phosphate dibasic salt, or mono-sulfate salt.  
     
     
         10 . The composition of  claim 9 , wherein said mono-phosphate dibasic salt is mono-phosphate dibasic salt of glycerol.  
     
     
         11 . The composition of  claim 9 , wherein mono-phosphate dibasic salt of glycerol is selected from the group consisting of glycerol-2-phosphate dibasic salt, sn-glycerol 3-phosphate dibasic salt and L-glycerol-3-phosphate dibasic salt.  
     
     
         12 . The composition of  claim 1 , wherein chitosan is further mixed with phosphate buffer and salt.  
     
     
         13 . The composition of  claim 1 , further comprising a biologically active factor.  
     
     
         14 . The composition of  claim 13 , wherein the biologically active factor is selected from the group consisting of cells, a hormones, a drug, DNA, a bulking agent, a growth factors, a DNA, DNA-polymer complex, liposomes, a pharmacological agent, a metabolic factor, an antibody, a nutritive factor, an angiogenic factor, and a radioisotope.  
     
     
         15 . The composition of  claim 14 , wherein said cells are live cells.  
     
     
         16 . The composition of  claim 14 , wherein the cells are nucleus pulpopus, annulus fibrosis, or a mixture thereof.  
     
     
         17 . The composition of  claim 14 , wherein the cells are embryonic stem cells or stem cells derived from a tissue selected from the group consisting of bone marrow, adipose, muscle, brain, skin, liver, vascular smooth muscle, endothelium, blood, or placenta.  
     
     
         18 . The composition of  claim 14 , wherein the cells are primary cells, differentiated cells, genetically modified cells, hybridomas, immortalized cells, transformed cells, tissue fragment cells, organelles, or a mixture thereof, nucleated cells, enucleated cells, germ cells, platelet cells, matrix vesicles, cell vesicles, demineralized bone paste, bone chips, cartilage fragments, or cell fragments or tissue fragments.  
     
     
         19 . The composition of  claim 14 , wherein the cells are autologous cells, allogeneic cells or xenogeneic cells.  
     
     
         20 . The composition of  claim 14 , wherein the biologically active factor is a cell attachment factor selected from the group consisting of fibrinogen, fibrin, fibronectin, hyaluronic acid, heparin, collagen, polylysine, polyornithine, receptor-binding cyclic peptide, receptor-binding protein.  
     
     
         21 . The composition of  claim 14 , wherein the biologically active factor is an enzyme, a growth-factor or a growth factor-immobilized substance.  
     
     
         22 . The composition of  claim 14 , wherein the biologically active factor is a plasmid DNA in the form of liposomes, a lipid complex, a chitosan complex, a poly-lysine complex, a DEAE dextran complex.  
     
     
         23 . The composition of  claim 14 , wherein the biologically active factor is a vaccine.  
     
     
         24 . The composition of  claim 23 , wherein the vaccine comprises an infective viral particle.  
     
     
         25 . The composition of  claim 14 , wherein the biologically active factor is a nutritive or metabolic factor.  
     
     
         26 . The composition of  claim 25 , wherein the a nutritive or metabolic factor is a lipid, amino acids, and a co-factor selected from the group consisting of cholesterol, glutamin, glucosamine, ascorbic acid, pyruvate, and lactate.  
     
     
         27 . The composition of  claim 14 , wherein the biologically active factor is at least one element selected from the group consisting of peripheral blood, bone blood, cord blood, a blood product, blood-borne cells, serum, platelets, platelet-rich plasma, fibrinogen, a clotting factor, and a blood-borne enzyme.  
     
     
         28 . The composition of  claim 14 , wherein the biologically active factor is an osteogenic substance.  
     
     
         29 . The composition of  claim 28 , wherein the osteogenic substance is a member of the bone morphogenetic protein family selected from the group consisting of TGF-p1, BMP-2, BMP-6, BMP-7, or a mixture thereof.  
     
     
         30 . The composition in  claim 1 , wherein hydroxyl-containing polymer is polyvinyl alcohol, dextran, linked with a bifunctional reactive aldehyde.  
     
     
         31 .- 40 . (canceled)  
     
     
         41 . A method for repairing soft tissue, said method comprising the step of administering the composition of  claim 1  at the site of a soft tissue in need of repair of a patient.  
     
     
         42 . A method for repairing or resurfacing a damaged cartilage, said method comprising the step of administering the composition of  claim 1  in or around a cartilage in need of repair or resurfacing of a patient.  
     
     
         43 . A method for repairing a meniscus of a patient, said method comprising the step of administering the composition of  claim 1  at the site of the meniscus in need of repair.  
     
     
         44 . The composition of  claim 3 , where the physiological medium comprises cell nutrients selected from the group consisting of glucose, amino acids, and vitamins, or a combination thereof, at isotonic and neutral pH.